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Hepat Mon. 2011;11(3):173-177

Milk thistle for treatment of nonalcoholic fatty liver disease

Ludovico Abenavoli 1*, Gabriella Aviello 2, Ra!aele Capasso 2, Natasa Milic 3, Francesco Capasso 2

1 Department of Experimental and Clinical Medicine, University of Magna Græcia, Catanzaro, Italy 2 Department of Experimental Pharmacology, University of Federico, Naples, Italy3 Department of Pharmacy, University of Novi Sad, Novi Sad, Serbia

c 2011 Kowsar M.P.Co. All rights reserved.

* Corresponding author at: Ludovico Abenavoli, Department of Experimental and Clinical Medicine, University of Magna Græcia, Viale Europa, Catanzaro, Italy. Tel: +39-9613697113, Fax: +39-961754220.

E-mail: l.abenavoli@unicz.it

A B S T R A C T

Nonalcoholic fatty liver disease (NAFLD) is one the most common causes of chronic liver dis-orders in the Western world. These patients have many significant comorbidities. The thera-peutic approach to NAFLD is based on lifestyle intervention, but there is no consensus on the ideal pharmacological treatment. Silybum marianum, commonly known as milk thistle (MT), is one of the oldest and most extensively researched plants in the treatment of liver diseases. Many studies have demonstrated that the active components of MT silymarin have many hepatoprotective properties. In recent years, several preclinical and clinical reports have described the e"cacy of silymarin as a treatment for NAFLD. The chief aim of this review is to discuss the newest and most promising applications of MT in the treatment of NAFLD.

A R T I C L E I N F O

Article history:Received: 25 Sep 2010Revised: 14 Jan 2011Accepted: 17 Jan 2011

Keywords:Milk thistleSilymarinNonalcoholic fatty liver diseaseFibrosisInsulin resistance

Article Type:Review Article

c 2011 Kowsar M.P.Co. All rights reserved.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is one the most common causes of chronic liver disorders in the Western world. These patients have many significant comorbidities (e.g., diabetes, hypothyroidism and metabolic syndrome) (1). Its incidence in adults and children is rising rapidly due to the current obesity and type 2 diabetes epidemics (2). It is a multifaceted metabolic disorder and is encountered in clinical practice by many health care specialists—from primary care physicians and gastroenterologists to cardi-ologists, radiologists, and gynecologists. The umbrella term “NAFLD” encompasses simple steatosis, nonalcoholic ste-atohepatitis (NASH), and advanced fibrosis or cirrhosis that is related to this pathological entity (3). The mechanism of the occurrence and progression of the underlying steatosis

Implication for health policy/practice/research/medical education: This article describes the importance of natural treatment regimen like plant extracts in treating NAFLD and can be attended by general practitioners and family physicians and others who are involved in treating patients with liver disorders.

Please cite this paper as: Abenavoli L, Aviello G, Capasso R, Milic N, Capasso F. Milk thistle for treatment of nonalcoholic fatty liver disease. Hepat Mon. 2011;11(3):173-177.

to liver disease is poorly understood but is likely driven by several factors that are expressed in the context of genetic predisposition. In this complex repertoire, a two-step hy-pothesis has been proposed, in which the first step induces the accumulation of liver fat and the second step e!ects the progression of steatosis to NASH (4, 5).

Obesity, insulin resistance, oxidative stress, and cytokine and adipokines mediate the pathogenesis of NAFLD. These factors can promote and enhance inflammation, cell in-jury, apoptosis, fibrinogenesis, and carcinogenesis, leading to the accumulation of fat, reflecting the development and progression of the disease. With regard to therapy, the ap-proach to NAFLD is based on lifestyle intervention, and there is no consensus on the ideal pharmacological treatment (6). Accordingly, weight reduction, regular physical activity, and insulin-sensitizing drugs have been used widely and exam-ined in several studies. Other treatment approaches include the consumption of special diets, antioxidants, and cytopro-tective therapy.

Silybum marianum, commonly known as milk thistle (MT)

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174 Abenavoli L et al. Milk thistle for treatment of NAFLD

(family Asteraceae/Compositae) is one of the oldest and most extensively studied plants in the treatment of liver diseases. This plant grows as a stout thistle in rocky soils, generating large purple flowering heads. Its leaves are characterized by milky veins, from which the plant derives its name (7). MT was used by ancient physicians and herbalists to treat liver and gallbladder disorders, including hepatitis, cirrho-sis, and jaundice, and to protect the liver against poisoning from chemical and environmental toxins, including snake bites, insect stings, mushroom poisoning, and alcohol. The active complex of MT is a lipophilic extract from its seeds and comprises three flavonolignan isomers, collectively known as silymarin. Silymarin acts as an antioxidant by re-ducing free radical production and lipid peroxidation and has antifibrotic activity, limiting the activation of hepatic stellate cells, inducing hepatic stellate cell apoptosis, and evoking the degradation of collagen deposits (8). In addi-tion, the ameliorative e!ects of silymarin in NAFLD patients might be attributed to its activity against glucose and lipid metabolism. Silymarin inhibits the activation of NF-kB and its related pathways in the liver. The principal aim of this review is to identify the newest and most promising applica-tions of MT in the treatment of NAFLD.

Biochemistry and pharmacology of milk thistle

The active extract of MT, known as silymarin, is a mixture of flavanolignans (Figure 1): silibinin, isosilibin, silidianin, and silichristine. Silymarin is extracted from dried MT seeds, in which it exists in higher concentrations than in other parts of the plant. The structural similarity of silymarin to steroid hormones is believed to mediate its protein synthesis facili-tatory actions. Silibin is the predominant and most active component, constituting approximately 60% to 70% of the isomers, followed by silichristin (20%), and silidianin (10%) (7, 8). Most of its hepatoprotective properties are attributed to silybin (silibinin), which is the chief constituent (60% to 70%) of silymarin (7, 8). Silymarin constitutes at least 70% of standardized milk thistle. It can be extracted with aqueous alcohol (95%) as a rich, bright yellow fraction. A hydroextrac-tion technique has also been developed to extract silymarin from MT (9). The silymarin content in milk thistle extracts can vary from 40% to 80% (8). The drug can be examined with regard to its microscopic characteristics by thin-layer chro-matography (TLC), high-performance liquid chromatogra-phy (HPLC), and spectrophotometry (10, 11).

Silymarin is insoluble in water and is typically admin-istered as a sugar-coated tablet or an encapsulated stan-dardized extract. Approximately 20% to 50% of silymarin is absorbed following oral administration in humans, and roughly 80% of the dose is excreted in bile, while about 10% enters enterohepatic circulation (11). Pharmacokinetic stud-ies, however, have been performed primarily using silibinin. The bioavailability of silibinin is low and appears to depend on several factors, such as (i) the content of accompanying substances that have solubilizing properties, such as other flavonoids, phenol derivates, amino acids, proteins, tocoph-erol, fat, cholesterol, and other substances that are found in the preparation; and (ii) the concentration of the prepara-tion itself (12). The systemic bioavailability can be enhanced by adding solubilizers to the extract (13).

The bioavailability of silybinin can also be increased by complexation with phosphatidylcholine or ß-cyclodextrin

and, possibly, by the choice of the capsule material (14). Phar-macokinetic studies on the silybin-phosphatidylcholine complex have demonstrated increased oral bioavailability of silybin in healthy human subjects, likely due to facilita-tion of the passage of the drug across the gastrointestinal tract by the drug complex (15). The variations in the content, dissolution, and oral bioavailability of silybinin between commercially available silymarin-containing products (de-spite the same declaration of content) are significant (16). Therefore, comparisons between studies should be made with caution, based on the analytical method (TLC vs. HPLC) and whether free, conjugated, or total silybinin is being measured. Systemic plasma concentrations are usually mea-sured—although silymarin is active in the liver—because they are an estimate of the quantity of the drug that is absorbed from the gastrointestinal tract. The adequate bioavailability accounts for the dose-related oral activity of silymarin in the liver (13-17).

In male volunteers, after a single administration of a standard dose of oral silibinin 100 to 360 mg, the Cmax of plasma silibinin was reached after approximately 2 hours and ranged between 200 and 1400 µg/L, of which approxi-mately 75% was present in conjugated form (15, 16, 18). The elimination half-life of total silibinin was approximately 6 hours (19, 20). Between 3% and 8% of the oral dose was ex-creted in the urine, and 20% to 40% was recovered from the bile as glucuronide and sulfate conjugates. The remainder was excreted in feces. Silibinin concentrations in the bile were approximately 100-fold higher than in the serum (10-5 to 10-4 mol/L of silibinin in bile), with concentrations peak-ing within 2 to 9 hours (19). At oral doses of 20 g/kg in mice and 1 g/kg in dogs, silymarin e!ects low toxicity and no mor-tality or adverse e!ects. After intravenous infusion, its LD50 was 400 mg/kg in mice, 385 mg/kg in rats, and 140 mg/kg in rabbits and dogs (21). Although silymarin has a good safety record, there are several reports of gastrointestinal distur-bances and allergic skin rashes with its use (22). These data demonstrate that the acute, subacute, and chronic toxicity of silymarin is very low.

Hepatoprotective e!ects of milk thistle

The active extract has antioxidant, anti-inflammatory, and antifibrotic properties; in addition, it stimulates protein biosynthesis and liver regeneration. There are four overarch-ing hepatoprotective activities of silymarin: (i) its e!ects against lipid peroxidation due to free radical scavenging and the ability to increase the cellular content of glutathi-one (GSH); (ii) its ability to regulate membrane permeability and increase membrane stability in the presence of xenobi-otic damage; (iii) its capacity to regulate nuclear expression through steroid-like e!ects; and (iv) its inhibition of the transformation of stellate hepatocytes into myofibroblasts, which mediate the deposition of collagen fibers, leading to cirrhosis (23-26). In addition, MT inhibits the absorption of toxins, such as phalloidin and -amanitin, preventing them from binding to the cell surface and inhibiting membrane transport systems. Further, by interacting with the lipid component of cell membranes, silymarin and silibinin can modulate their chemical and physical properties. They stabi-lize the membranes of hepatocytes and thus prevent toxins from entering them from enterohepatic circulation. They promote liver regeneration by stimulating nucleolar poly-

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175Abenavoli L et al.Milk thistle for treatment of NAFLD

merase A and increasing ribosomal protein synthesis (27). Silymarin inhibits the expression of adhesion molecules, such as E-selectin, another family of transmembrane mol-ecules, which are expressed preferentially on the surface of leukocytes (28). Its hepatoprotective properties against a wide range of liver damage-inducing agents render MT a unique drug.

Milk thistle in liver steatosis

Several well-designed experimental studies have suggest-ed that silymarin exerts beneficial e!ects in chronic liver diseases, particularly in NAFLD (Figure 2). For example, sily-marin interferes with leukotriene formation in Kup!er cell (KC) cultures, thus inhibiting hepatic stellate cell (HSC) acti-vation, a crucial event in fibrogenesis (26). In addition, 10-4 mol/l silymarin blocks the proliferation of HSC cultures and their transformation into myofibroblasts (29). Velussi et al. (30) studied the e"cacy of silymarin in reducing lipid per-oxidation and insulin resistance in diabetic patients with alcoholic cirrhosis. The study was performed in alcoholic cirrhosis patients, who have similar natural histories and pathological features as alcoholic liver disease and NASH pa-tients. In this randomized, controlled, unblinded, 12-month study, one group (n = 30) received 600 mg silymarin per day plus standard therapy, and the control group (n = 30) received standard therapy alone. The e"cacy parameters, measured regularly throughout the study, included fasting blood glucose levels; mean daily blood glucose levels, daily glycosuria levels, glycosylated hemoglobin (HbA1c), and ma-londialdehyde (MDA) levels, a marker of lipid peroxidation.

There was a significant decrease (p < 0.01) in fasting blood

glucose levels, mean daily blood glucose levels, daily gly-cosuria, and HbA1c levels after 4 months of treatment with silymarin. Moreover, fasting insulin levels and mean exog-enous insulin requirements declined in the treated group (p < 0.01), and the control group experienced an increase (p < 0.05) in fasting insulin levels and stabilized their need for in-sulin. These findings were consistent with the significant de-crease (p < 0.01) in basal and glucagon-stimulated C-peptide levels in the treated group and the rise in both parameters in the control group. Notably, MDA levels fell in the treated group (p < 0.01). These studies demonstrate that treatment with silymarin reduces lipoperoxidation of cell membranes and insulin resistance, decreasing the overproduction of endogenous insulin and the need for exogenous insulin sig-nificantly.

Subsequently, Loguercio et al. (31) evaluated the antioxi-dant and antifibrotic activity of a complex that comprised silybin, vitamin E, and phospholipids (Realsil ® IBI-Lorenzi-

Figure 1. Structures of the components of silymarin.

Figure 2. Pathogenic mechanisms in the histological progression of NAFLD and the site of action of sylimarin (crossed circle) (CYP2E1: cytochrome P450 2E1, ROS: react-ive oxygen species, HSCs: hepatic stellate cells, KC: Kup!er cells)

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176 Abenavoli L et al. Milk thistle for treatment of NAFLD

ni Pharmaceutical, Italy) against insulin resistance and liver damage in patients with NAFLD and chronic HCV infection. This study enrolled 85 patients; 59 were a!ected by primitive NAFLD (group A), and 26 had HCV-related chronic hepatitis C with NAFLD, all HCV genotype-1b, and non-responders to the previous antiviral treatment (group B). All patients with a diagnosed liver disease in the 2 years prior to the study, based on histological criteria, were enrolled over 6 consecu-tive months and subdivided using a systematic random sampling procedure: 53 patients (39 NAFLD and 14 HCV) were treated with 4 tablets/day of Realsil ® (one tablet contained 94 mg of silybin, 194 mg of phosphatidylcholine, and 90 mg of vitamin E) for 6 months, followed by another 6 months of follow-up, and 32 patients (20 NAFLD and 12 HCV) consti-tuted the control group (no treatment).

At 0, 6, and 12 months, the following outcomes were mea-sured: body mass index (BMI), bright liver by ultrasonogra-phy (US), transaminase and GGT levels, blood glucose and insulin plasma levels with simultaneous measurement of in-sulin resistance by Homeostasis Model Assessment (HOMA) test, and plasma levels of transforming growth factor ß, hy-aluronic acid and metalloproteinase as indices of liver fibro-sis. Group A showed a significant and persistent reduction in US score for liver steatosis that ranged p < 0.01. Plasma levels of liver enzymes fell in treated patients but not in the control group, but this e!ect lasted only in NAFLD patients. Hyperinsulinemia, present in both groups, declined only in treated patients (p < 0.005). Realsil ® significantly reduced all indices of liver fibrosis in both treatment groups, persist-ing only in group B.

In a randomized clinical trial, Hajaghamohammadi et al. (32) examined the e"cacy of silymarin in 50 NAFLD pa-tients. The study population, comprising 32 men (64%) and 18 women (36%), was divided into case and control groups. All patients had elevated liver enzymes and increased liver echogenicity by US. The case group was treated with one tab-let that contained 140 mg silymarin per day for 2 months; the control group was treated similarly with placebo. Before and after the study, weight, BMI, and liver transaminase levels were measured for each patient. The authors did not observe any significant di!erences in mean weight or BMI before or after the study in either group. In the case group, mean alkaline transaminase (ALT) and aspartate transami-nase (AST) levels deceased from 103.1 to 41.4 U/L and 53.7 to 29.1 IU/ml, respectively (p < 0.001 and p < 0.001, respective-ly). In the control group, the decreases in mean ALT and AST (7.8 and 2.2 IU/ml respectively) were not significant.

The e!ect of silymarin on transaminase levels was con-firmed by another Iranian study (33). One hundred subjects with NASH were randomized into two groups: group A, com-prising 29 males and 21 females, received placebo, and group B, with 28 males and 22 females, received 280 mg silymarin for 6 months. The mean serum ALT level in the silymarin group was 113.03 and 73.14 IU/ml before and after the treat-ment, respectively (p = 0.001). ALT normalization (ALT < 40) was observed in 18% and 52% of patients in groups A and B, respectively (p = 0.001). AST normalization (AST < 40) was observed in 20% of cases in the placebo group and in 62% of cases in the silymarin-treated group (p = 0.0001). Mitochon-dria regulate hepatocyte metabolism, constituting the site of ß-oxidation and oxidative phosphorylation. Oxidative stress in NASH is closely related to mitochondrial dysfunc-tion (34). During the progression of NASH, the excess of free

fatty acids increases mitochondrial H#O# production, which in turn oxidizes mitochondrial membranes and regulates the activity of uncoupling protein 2 (UCP2) and carnitine palmitoyl transferase-1 (CPT-1) (35).

Serviddio et al. (36) examined the e!ects of the silybin-phos-pholipid complex on liver redox balance and mitochondrial function in a dietary model of NASH, measuring glutathione oxidation, mitochondrial oxygen uptake, proton leak, ATP homeostasis, and H2O2 production rate in liver mitochon-dria from rats that were fed a methionine/choline-deficient diet (MCD) and MCD plus SILIPHOS for 7 and 14 weeks. Oxi-dative proteins, hydroxynonenal (HNE) - and MDA-protein adducts, and mitochondrial membrane lipid composition were also assessed. SILIPHOS limited glutathione deple-tion and mitochondrial H2O2 production. Moreover, this complex preserved mitochondrial bioenergetics and pre-vented mitochondrial proton leakage and ATP reduction. The silybin-phospholipid complex limited the formation of HNE- and MDA-protein adducts. In conclusion, this complex prevents severe oxidative stress and preserves hepatic mito-chondrial bioenergetics in MCD-induced NASH. The altera-tions in mitochondrial membrane fatty acid composition that were induced by the MCD diet were prevented in part by silybin and phospholipids, which conferred anti-inflam-matory and antifibrotic e!ects.

Recently Haddad et al. (37) examined the therapeutic e!ect of silibinin in an experimental rat model of NASH. The con-trol group was fed a standard liquid diet for 12 weeks, and the test animals were fed a high-fat liquid diet for 12 weeks with or without (NASH) a daily supplement of silibinin-phosphatidylcholine complex (silibinin 200 mg/kg) for the last 5 weeks. The NASH rats developed all hallmarks of the pathology. Treatment with silibinin improved liver steatosis and inflammation and decreased lipid peroxidation, plas-ma insulin, and TNF-alpha (p<0.05). In addition, silibinin decreased the release of free radicals and restored relative liver weights and GSH levels (p<0.05). The authors conclud-ed that a complex with phosphatidyl-choline is e!ective in reversing steatosis, inflammation, oxidative stress, and insu-lin resistance in an in vivo rat model of diet-induced NASH.

Conclusions

NAFLD and its various stages a!ect much of the world's population. The pathogenic mechanisms of liver damage that are involved in NAFLD are complicated and comprise a series of sequential steps. With regard to therapy, the ap-proach to NAFLD is currently based on lifestyle intervention, but there is no consensus on the ideal pharmacological treatment (2). The drugs that are used to treat NAFLD should reduce body weight, improve insulin resistance and other metabolic alterations, reduce the link between adipose tis-sue and liver function by acting as anti-inflammatory and immunomodulatory agents, and modulate the progression of liver steatosis to inflammation and fibrosis by blocking oxidative stress. A multifaceted approach to NAFLD, entail-ing several treatment options, is likely to be developed soon. Among these strategies, the use of complementary and al-ternative medicines, such as natural antioxidants and he-patoprotective plant products, has been widely accepted in the past decade. Silymarin is one of the most successful examples of a modern drug that arose from traditional heal-ing practices. It is favored in treating various liver diseases

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177Abenavoli L et al.Milk thistle for treatment of NAFLD

due to its oral e"cacy, good safety profile, and, most impor-tantly, a!ordability.

Several pharmacological studies have been performed on the active components of MT, silymarin, and silibinin. These substances have hepatoprotective, antioxidant, anti-inflammatory, and antifibrotic properties; in addition, they stimulate protein biosynthesis and liver regeneration and have immunomodulatory activity (7). Particularly with re-gard to NAFLD patients, the ameliorative e!ects of silybin in diabetic patients, due to improved insulin activity, reduc-tions in lipid peroxidation, and restoration of GSH levels, might explain its e"cacy against liver steatosis (31, 38). Based on the literature, we believe that MT is a useful medicinal herb that is a viable therapeutic option for treating patients with NAFLD.

Finantial support

None declared.

Conflicts of Interest

The authors have declared that there is no conflict of inter-

est.

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